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| archels | .tell alu re your trans vs. posthuman question, if you want a thorough answer you should read Stefan Sorgner's latest book | 02:47 |
|---|---|---|
| yoleaux | archels: I'll pass your message to alu. | 02:47 |
| archels | but I side with the rest of the channel on this one: it's a very non-universal and somewhat pointless distinction | 02:47 |
| archels | everyone has their own ideas of what these terms "mean" | 02:47 |
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| -!- Topic for ##hplusroadmap: biohacking, nootropics, transhumanism, open hardware | sponsored by lobsters everywhere, banned by the Federal Death Administration (5 times) | this channel is LOGGED: http://gnusha.org/logs | http://diyhpl.us/wiki | "ray kurzweil is a pessimist" - george church | 05:59 | |
| -!- Topic set by kanzure [~kanzure@unaffiliated/kanzure] [Wed May 20 12:46:25 2015] | 05:59 | |
| [Users ##hplusroadmap] | 05:59 | |
| [ Acty ] [ CaptHindsight] [ EnabrinTain ] [ kanzure ] [ Porb_ ] [ Stskeeps ] | 05:59 | |
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| -!- Irssi: ##hplusroadmap: Total of 83 nicks [0 ops, 0 halfops, 0 voices, 83 normal] | 05:59 | |
| !asimov.freenode.net [freenode-info] if you're at a conference and other people are having trouble connecting, please mention it to staff: http://freenode.net/faq.shtml#gettinghelp | 05:59 | |
| -!- Channel ##hplusroadmap created Thu Feb 25 23:40:30 2010 | 05:59 | |
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| kanzure | yes i'm not sure there's any benefit conferred by one versus the other term | 06:26 |
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| JayDugger | Good morning, everyone. | 06:40 |
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| dao | hi | 06:55 |
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| kanzure | hello dao | 07:15 |
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| wrldpc1 | dao .. as in 道？ | 07:52 |
| archels | the dao that can be spoken is not the eternal dao | 07:53 |
| fenn | if you meet the dao on the road, kill him | 07:55 |
| kanzure | do i have to | 07:55 |
| fenn | .wik dao vallis | 07:56 |
| yoleaux | "Dao Vallis is a valley on Mars that appears to have been carved by water. It runs southwestward into Hellas Planitia from the southern slopes of the volcano Hadriacus Mons, and has been identified as an outflow channel. It and its tributary, Niger Vallis, extend for about 1,200 km (750 mi)." — https://en.wikipedia.org/wiki/Dao_vallis | 07:56 |
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| fenn | it's also a number in hexadecimal, and a color i use a lot | 07:57 |
| archels | hey, that's not a bad looking colour | 07:59 |
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| fenn | .wik decentralized autonomous organization | 08:06 |
| yoleaux | "A decentralized autonomous organization (DAO), fully automated business entity (FAB), or distributed autonomous corporation/company (DAC) is a decentralized network of narrow-AI autonomous agents which perform an output-maximizing production function and which divides its labor into computationally intractable tasks (which it incentivizes …" — https://en.wikipedia.org/wiki/Decentralized_autonomous_organization | 08:06 |
| fenn | please don't actually bother with any of those links | 08:08 |
| kanzure | as far as i can tell the primary interest that people have in "decentralized autonomous organizations" is using the concept as a method to skirt business registration and regulation | 08:11 |
| kanzure | in the posam operations document there's a lot of steps that involve non-synthesizer equipment, like a rotary mixer apparatus. ugh. | 08:26 |
| kanzure | i'm also a little confused why they had only one humidity sensor. wouldn't you want one on each far corner of the gas-enclosure chamber? | 08:27 |
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| kanzure | pre-synthesis script http://diyhpl.us/~bryan/papers2/DNA/posam/pogo/pogo-vb/Basic.pp1 | 08:36 |
| kanzure | stepwise synthesis script http://diyhpl.us/~bryan/papers2/DNA/posam/pogo/pogo-vb/Basic.pp2 | 08:37 |
| kanzure | post-synthesis script (just flushing) http://diyhpl.us/~bryan/papers2/DNA/posam/pogo/pogo-vb/Basic.pp3 | 08:37 |
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| kanzure | huh i don't think they were doing a specific oligo sequence | 08:45 |
| kanzure | ah nevermind | 08:48 |
| kanzure | "Dim iBank(6) As Integer 'E.g. 1:X/88 2:Y/89 3:A/65 4:C/67 5:G/71 6:T/84." | 08:48 |
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| CaptHindsight | the POSaM has the sample tray mounted and moving on a X-axis positioner and the printhead on a ZY combo | 09:14 |
| CaptHindsight | what is our target accuracy and repeatability of each axis? | 09:15 |
| kanzure | well we need to do up to 80 or 100 layers per drop | 09:16 |
| CaptHindsight | yesterdays discussion mentioned that variances in drop placement will have an effect on synthesis | 09:16 |
| kanzure | and probably the head has to be moved long-distance during purge steps, so whatever it takes to get it back to the correct location | 09:16 |
| CaptHindsight | Epson doesn't have a public spec on drop variation for volume or straightness | 09:18 |
| kanzure | i think this is just an optimization issue | 09:18 |
| CaptHindsight | most piezo heads hold +/- 10% drop size variation and straightness is <1.5 deg from centerline | 09:19 |
| CaptHindsight | since the sample trays are non-porous the actual drop size will vary based on the fluids surface tension and the surface tension of the surface it lands on | 09:20 |
| CaptHindsight | drop size = landed drop diameter/size of splotch | 09:21 |
| CaptHindsight | so the fluids and reactions near the outer edge of the drops will have the most variance | 09:23 |
| CaptHindsight | bbl | 09:23 |
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| eudoxia | who wants five bucks in starbucks credit | 10:18 |
| eudoxia | a client gave it to me and we don't have starbucks in pooruguay | 10:18 |
| kanzure | you let someone pay you $5? | 10:23 |
| eudoxia | they asked me for feedback on a tiny app and i gave them a five minute email to ~spread the good vibes~, technically it was work-related so i got paid | 10:24 |
| CaptHindsight | the POSaM papers don't mention anything about tweaking drive waveforms since they can't modify the fluids to adjust for viscosity, surface tension etc | 10:35 |
| CaptHindsight | it looks like they just got lucky | 10:35 |
| nmz787_i1 | most biologists probably wouldn't know about drive waveforms | 10:36 |
| nmz787_i1 | unless the off-the-shelf controller supported it | 10:36 |
| CaptHindsight | it's impressive that they did as much as they did | 10:36 |
| nmz787_i1 | (for stepper controllers the most I've seen is mixed decay mode) | 10:36 |
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| kanzure | .title http://arxiv.org/abs/1504.07065 | 10:48 |
| yoleaux | [1504.07065] The doubly eclipsing quintuple low-mass star system 1SWASP J093010.78+533859.5 | 10:48 |
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| kanzure | .wik glymphatic system | 11:40 |
| yoleaux | "The glymphatic system (or glymphatic clearance pathway) is a functional waste clearance pathway for the mammalian central nervous system (CNS)." — https://en.wikipedia.org/wiki/Glymphatic_system | 11:40 |
| kanzure | .title http://www.sciencemag.org/content/290/5490/350 | 11:41 |
| yoleaux | Replaying the Game: Hypnagogic Images in Normals and Amnesics | 11:41 |
| kanzure | .title http://www.sciencemag.org/content/342/6156/373 | 11:41 |
| yoleaux | Sleep Drives Metabolite Clearance from the Adult Brain | 11:41 |
| kanzure | "Impairment of paravascular clearance pathways in the aging brain" http://www.researchgate.net/profile/Benjamin_Kress/publication/236601893_Evaluating_glymphatic_pathway_function_utilizing_clinically_relevant_intrathecal_infusion_of_CSF_tracer/links/54e265980cf2edaea092e8f2.pdf | 11:43 |
| kanzure | hmph | 11:56 |
| kanzure | "There is some supporting evidence of the restorative function of sleep. The sleeping brain has been shown to remove metabolic waste products at a faster rate than during an awake state.[88] While awake, metabolism generates reactive oxygen species, which are damaging to cells. In sleep, metabolic rates decrease and reactive oxygen species generation is reduced allowing restorative processes to take over. It is theorized that sleep helps ... | 11:56 |
| kanzure | ... facilitate the synthesis of molecules that help repair and protect the brain from these harmful elements generated during waking.[89] The metabolic phase during sleep is anabolic; anabolic hormones such as growth hormones (as mentioned above) are secreted preferentially during sleep. The duration of sleep among species is, broadly speaking, inversely related to animal size[citation needed] and directly related to basal metabolic rate ... | 11:56 |
| kanzure | ... (BMR). Rats, which have a high BMR, sleep for up to 14 hours a day, whereas elephants and giraffes, which have lower BMRs, sleep only 3–4 hours per day." | 11:56 |
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| drethelin | wasn't there a study recently about a specific chemical in the brain that was removed during sleep | 12:01 |
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| kanzure | another crispr overview http://andrew.gibiansky.com/blog/genetics/crispr/ | 12:19 |
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| CaptHindsight | kanzure: http://www.fujifilmusa.com/products/industrial_inkjet_printheads/deposition-products/dmp-2800/ $30k off the shelf | 13:26 |
| kanzure | friend of mine says she was working with this one http://www.fujifilmusa.com/shared/bin/PDS00085-DMP2831.pdf | 13:27 |
| CaptHindsight | thats it | 13:27 |
| kanzure | but she was cheating, she says she just went to their offices to use it (computer had a driver installed on it) | 13:27 |
| kanzure | oh well | 13:27 |
| CaptHindsight | i think they charge 1.5K/day for lab use | 13:28 |
| kanzure | yea she's part of some research group | 13:28 |
| kanzure | so either paid or research collaboration project thingy | 13:28 |
| CaptHindsight | should we stay at 25um repeatability or tighter? | 13:30 |
| kanzure | let's aim for maybe a milion spots ish? so i think the repeatability and spacing have some relationship that i forget. | 13:31 |
| kanzure | actually i don't know how many spots to aim for | 13:31 |
| kanzure | at least a million. but if we can manage way more, then why not.. | 13:32 |
| CaptHindsight | POSam had ~150um dia spots | 13:33 |
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| delinquentme | kanzure, do you have access to JOVE? | 13:33 |
| delinquentme | nmz787, fenn | 13:34 |
| kanzure | not at the moment | 13:34 |
| kanzure | delinquentme: http://diyhpl.us/~bryan/jove.urls.txt | 13:34 |
| kanzure | they have since moved their files though :-( | 13:34 |
| CaptHindsight | run the numbers, how many spots, what spot dia and what spacing? | 13:35 |
| delinquentme | http://www.jove.com/video/50262/design-and-use-of-multiplexed-chemostat-arrays | 13:35 |
| delinquentme | derp. | 13:35 |
| delinquentme | http://www.jove.com/video/50168/the-use-of-chemostats-in-microbial-systems-biology | 13:35 |
| delinquentme | that one | 13:35 |
| kanzure | CaptHindsight: spot diameter should be based on giving a "good margin for error" or something- e.g., something not at the max performance of the poor inkjet head | 13:35 |
| kanzure | CaptHindsight: additionally, it needs to be based on the micropipetting that will be necessary for combining different spots | 13:35 |
| CaptHindsight | it looks like the surface tension of their tray limited their drop density | 13:36 |
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| kanzure | oh | 13:36 |
| CaptHindsight | how many layer max are we shooting for? | 13:37 |
| kanzure | well, because of chemistry yield errors, i think it's <100 bp, probably <80 bp | 13:37 |
| CaptHindsight | maybe we should SLA print sample trays with wells | 13:38 |
| kanzure | 50 would be nice... it would give us 20 bp on both sides for dna hybridization, and then 10 bp of unique content | 13:38 |
| CaptHindsight | maybe 1mm deep wells | 13:39 |
| kanzure | the wells could be wider than the drops- which might be favorable for splash reasons, or surface tension issues, etc.. | 13:39 |
| CaptHindsight | they counted on surface tension to keep the drops from spreading | 13:39 |
| kanzure | oh ok | 13:40 |
| CaptHindsight | we could use a photopolymer | 13:40 |
| kanzure | there have been some photopolymer methods of dna synthesis | 13:40 |
| CaptHindsight | print the walls of the wells | 13:41 |
| kanzure | http://diyhpl.us/~bryan/papers2/DNA/Light-directed%20synthesis%20of%20high-density%20oligonucleotide%20arrays%20using%20semiconductor%20photoresists%20-%201996.pdf | 13:41 |
| CaptHindsight | yeah, V2 | 13:41 |
| kanzure | hehe they had dna densities of 10^6 strands per cm^2 | 13:41 |
| CaptHindsight | their limit was 90 dpi | 13:41 |
| ParahSailin_ | i got a bunch of azure bizspark capacity to waste | 13:45 |
| ParahSailin_ | maybe i should stuff it full of video | 13:46 |
| kanzure | get the 1000genomes data set | 13:46 |
| kanzure | make some indexes etc | 13:46 |
| ParahSailin_ | oh thats not a bad idea | 13:46 |
| ParahSailin_ | sra is such a bizarre file format | 13:47 |
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| ParahSailin_ | but ncbi tools work on them about 75% of the time | 13:47 |
| ParahSailin_ | the 25% is really awful | 13:47 |
| kanzure | is that their format for 1000potatoes? | 13:47 |
| ParahSailin_ | doesnt azco use electrochemical control for their array synth? | 13:47 |
| ParahSailin_ | yeah, short read archive | 13:48 |
| ParahSailin_ | admittedly sra uses some pretty clever compression | 13:48 |
| CaptHindsight | will WASH WITH ACETONITRILE be able to properly dry if we have shallow wells? | 13:48 |
| ParahSailin_ | but it is completely inpenetrable with any sort of third party tool | 13:48 |
| kanzure | ParahSailin_: captain is going to be making an oligo assembler, make lots of advice comments or else | 13:48 |
| ParahSailin_ | if someone gives me $20k i will make a usable third party library in python and c for dealing with sra files | 13:49 |
| ParahSailin_ | theoretically, ncbi has an sdk for sratoolkit, but it is totally abstruse | 13:50 |
| ParahSailin_ | maybe its intrinsic to the sra file format, similar to pdf, that makes it impossible to write good tools for | 13:50 |
| delinquentme | directed evolution kanzure ... surely weve got someone in here whos totally bad ass at this right? | 13:59 |
| nmz787_i | i'm badass at this, but not that | 14:00 |
| nmz787_i | CaptHindsight: won't shallow wells help evap? | 14:01 |
| nmz787_i | isn't evap same as dry? | 14:01 |
| kanzure | delinquentme: i know various things about the subject. state your request. | 14:05 |
| delinquentme | site directed mutagenesis ... this can be reduced to a liquid handling operation right? | 14:07 |
| delinquentme | plus heating / cooling | 14:07 |
| delinquentme | seems to be the case http://kirschner.med.harvard.edu/files/protocols/Stratagene_quickchangepdf.pdf | 14:09 |
| delinquentme | possibly bad assumption: all site directed mutagenesis protocols are like the above | 14:09 |
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| nmz787_i | delinquentme: crispr could probably be considered part of a newer strategy | 14:12 |
| nmz787_i | # flux ╨ | 14:14 |
| nmz787_i | # ╥ | 14:14 |
| nmz787_i | ah, damn, the extended ASCII doesn't show up well in the logs | 14:14 |
| kanzure | delinquentme: there's also things like http://arep.med.harvard.edu/pdf/Bonde_2014.pdf | 14:18 |
| delinquentme | kanzure, what would you guess is the time utilization on churches MAGE ? | 14:19 |
| kanzure | no idea | 14:22 |
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| kanzure | ParahSailin_: what's the ideal output of the dna synthesis machine? at first we're shipping the output to someone to do dna sequencing. but it's technically not dna yet. and also the physical format... they are not going to like 100 microscope slides with 10 million spots... | 15:22 |
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| kanzure | "he destruction of extracellular matrix by enzymes such as serine proteases, threonine proteases, and matrix metalloproteinases.[3][7]" | 15:43 |
| nmz787_i | kanzure: why would it not technically be DNA as output? | 15:45 |
| kanzure | it's just an oligo | 15:46 |
| kanzure | i don't even think it qualifies as ssDNA | 15:46 |
| ParahSailin_ | is it a nucleic acid? | 15:47 |
| kanzure | oh maybe it does qualify as ssDNA, weird | 15:48 |
| kanzure | well anyway, don't these places have requirements like you gotta pellet it, you have to deliver it on a specific tray? | 15:48 |
| ParahSailin_ | sequencers are used to getting whatever | 15:49 |
| kanzure | but surely not 1 million spots with 20 micron spacing? | 15:50 |
| ParahSailin_ | that would be pretty unusual | 15:50 |
| kanzure | :-) | 15:50 |
| kanzure | right, so we have to pick something that is normal and expected | 15:50 |
| ParahSailin_ | you wanna pool it all together and attach illumina tags? | 15:50 |
| ParahSailin_ | and dump it on a lane? | 15:51 |
| ParahSailin_ | that would work | 15:51 |
| kanzure | well... it would be nice to do microarray sequencing? | 15:51 |
| ParahSailin_ | you could even synthesize with illumina tags on both ends | 15:51 |
| nmz787_i | microarray is tag/affinity-binding based | 15:52 |
| kanzure | wait no, surely someone is doing microarray sequencing-by-synthesis with polymerase? | 15:52 |
| nmz787_i | yeah, that one company | 15:52 |
| ParahSailin_ | illumina is kinda | 15:52 |
| nmz787_i | not pacbio | 15:52 |
| nmz787_i | helix---something? | 15:52 |
| nmz787_i | helicos? | 15:52 |
| kanzure | blah | 15:53 |
| kanzure | ok, so maybe gibson assembly first, then ship a pellet to someone | 15:53 |
| ParahSailin_ | the new illumina flow cells look more like arrays with metered wells | 15:53 |
| ParahSailin_ | do what i said | 15:53 |
| kanzure | if you put it all in one lane, how would you know which sequence was wrong? | 15:53 |
| ParahSailin_ | attach illumina tags and send it in as a library | 15:53 |
| ParahSailin_ | approximate string matching | 15:54 |
| kanzure | illumina tags seem to be limited to 96 unique molecules at a time | 15:54 |
| ParahSailin_ | nah | 15:54 |
| kanzure | http://www.illumina.com/documents/products/datasheets/datasheet_sequencing_multiplex.pdf | 15:54 |
| ParahSailin_ | ideally when you do library prep you want ever cluster to be a unique molecule | 15:55 |
| kanzure | i am going to have 1 million unique molecules (or more) | 15:55 |
| kanzure | er.. 1 million different sequences. | 15:55 |
| ParahSailin_ | if you have a million unique sequences, thats what you will read out | 15:55 |
| kanzure | so i'll have a million unique illumina tags too? | 15:56 |
| kanzure | "each index is six bases in length" | 15:56 |
| ParahSailin_ | youd put a common illumina tag, and you could put a million random sequences along with your business biology if you wanted | 15:56 |
| ParahSailin_ | but i would think the biological sequence you want would be enough | 15:56 |
| ParahSailin_ | eh, we use 15mer indexes here at affy | 15:57 |
| kanzure | the point is to verify that the right thing was sequenced in each location/spot. it's the mapping between spot and unique dna molecule that is important. i suppose you could say "well just make sure none of your sequences are anywhere close to similar"... | 15:57 |
| kanzure | i was thinking of situations like "500 of the unique molecules are only unique by one nucleotide" | 15:58 |
| ParahSailin_ | make some unique tag for each then | 15:58 |
| kanzure | hm | 15:58 |
| kanzure | yeah i see what you mean now | 15:58 |
| kanzure | and this is the typical <$1000/run sequencing? | 15:59 |
| ParahSailin_ | i see what you're saying that it would be good to have a known mapping to location... but what are the odds that at well A1, you tell the machine to make ACGT and it makes GGGG instead | 15:59 |
| kanzure | well... who knows. myabe the machine sucks? | 15:59 |
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| ParahSailin_ | so that it swaps it with some other location | 15:59 |
| nmz787_i | in that case the tag wont even be right | 15:59 |
| ParahSailin_ | yeah more likely you will have no discernable read at all | 16:00 |
| kanzure | i wonder if we could do sequencing-by-polymerase on this machine. that might help. hmm. | 16:00 |
| ParahSailin_ | if you just synthesize your million spots so that they have Nmers with sufficient edit distance to each other, then you have a pretty reliable way to test the machine | 16:01 |
| kanzure | so what's the format that illumina shops would expect? just pipette everything into a tube and call it quits? | 16:01 |
| kanzure | yes, i agree | 16:01 |
| ParahSailin_ | stick it in a tube | 16:01 |
| kanzure | i wasn't assuming the edit distance thing earlier, but yes it makes sense to do it that way :-) | 16:01 |
| ParahSailin_ | how do you think they came up with the 6 base or 15 base index barcodes | 16:02 |
| kanzure | no idea | 16:02 |
| ParahSailin_ | illumina's 6 base ones were geared towards hamming distance | 16:03 |
| ParahSailin_ | which kinda sucks when you consider like index 3 and index 26 or something ended up being one indel apart, oops | 16:03 |
| kanzure | and their 15 bp index was not based on hamming distance? | 16:04 |
| ParahSailin_ | illumina doesnt have a 15bp index | 16:05 |
| ParahSailin_ | but basically anyone who uses their machines can make oligos that will read the same way as illumina's so they can make whatever indexes they like | 16:06 |
| kanzure | how do people normally do microarray to single tube? | 16:07 |
| kanzure | i can possibly arrange for there to be beads in each well/pore on the surface, and then just dump all the beads out at the end into a tube, then cut the dna off? | 16:07 |
| ParahSailin_ | add a drop of water and suck it back up | 16:07 |
| kanzure | or does everyone just do lots of micropipetting? | 16:07 |
| kanzure | maybe i can use a piece of tissue paper to absorb all the liquid, then dissolve the tissue paper and keep the dna | 16:09 |
| ParahSailin_ | what i said above is how we dealt with microarray synthesized oligos that we just needed to pool | 16:10 |
| kanzure | used a pipette to suck up each well? | 16:10 |
| ParahSailin_ | its not a well, its a glass slide | 16:10 |
| kanzure | that's a lot of pipetting if it's 1 million pores | 16:10 |
| kanzure | well whatever- i haven't decided whether to use glass slides or pores yet | 16:10 |
| ParahSailin_ | you add like one big drop of water and suck it up | 16:10 |
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| kanzure | oh, all of it at once. cool. | 16:11 |
| kanzure | does gibson work with pooling arbitrarily large sets? | 16:12 |
| ParahSailin_ | oh if you put any enzyme in that pot, it would make a whole bunch of junk | 16:13 |
| ParahSailin_ | much less three enzymes together | 16:13 |
| kanzure | why don't we have one-pot ligation techniques yet? bleh | 16:14 |
| kanzure | what would be a good assembly approach for 1 to 10 million unique strands? | 16:15 |
| ParahSailin_ | ligase is one enzyme, i think you could try that | 16:15 |
| ParahSailin_ | ive seen papers where they use one ligase and lots of ssdna | 16:15 |
| kanzure | also assume i can do 20 bp wings on both ends if necessary | 16:16 |
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| kanzure | .title http://www.nature.com/articles/nmeth.1318 | 16:20 |
| yoleaux | Access : Enzymatic assembly of DNA molecules up to several hundred kilobases : Nature Methods | 16:20 |
| kanzure | ah right, yeast assembly too | 16:20 |
| kanzure | gibson assembly, yeast assembly, golden gate, dna ligase, ligase cycling reaction, what's the one with dna hybridization and polymerase (just pcr assembly?), paperclip, golden braid, ... | 16:23 |
| kanzure | .title http://www.nature.com/articles/srep11302 | 16:23 |
| yoleaux | RapGene: a fast and accurate strategy for synthetic gene assembly in Escherichia coli : Scientific Reports : Nature Publishing Group | 16:23 |
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| nmz787_i | kanzure: see, on-chip sequencing makes everything easier | 16:49 |
| nmz787_i | and if it's on-chip, you don't even need sequencing as much as length-assertion | 16:49 |
| CaptHindsight | the POSaM papers leave out lots of little details | 16:51 |
| CaptHindsight | they mention the 90dpi but not the spot diameters they were getting | 16:52 |
| CaptHindsight | the 700 series heads were specified by Epson at 6pL but they were getting closer to 10pL drops... | 16:55 |
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| CaptHindsight | I don't know if that is because of the fluids that they were using or the waveform they chose | 16:55 |
| CaptHindsight | I don't know why they said that they were getting close to 10pL either, they don't say how they determined that | 16:56 |
| CaptHindsight | 1.000 drops into a microbalance or just a hunch | 16:57 |
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| kanzure | they may not have been measuring their spot diameter | 17:16 |
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| ParahSailin_ | you guys should have bought the GA2x if you wanted to reverse engineer chip sequencing | 18:12 |
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| ParahSailin_ | actually, my friend may have just stashed it in his warehouse | 18:13 |
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| CaptHindsight | since the Epson heads are specifically made for deposition but as low cost aqueous ink printers we'll just make the best of them | 18:17 |
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| CaptHindsight | sorry are not made for deposition | 18:17 |
| ParahSailin_ | kanzure: an azco array synthesis run is like a few k | 18:18 |
| ParahSailin_ | kanzure: i'd say start from there | 18:18 |
| ParahSailin_ | do an illumina library to see what the error rate is like | 18:19 |
| ParahSailin_ | then if its good enough look at whatever patents they have a rip them off | 18:22 |
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| kanzure | http://www.customarrayinc.com/oligos_main.htm | 19:41 |
| kanzure | 79 bp * 12472 for $1600 | 19:41 |
| kanzure | and 170 bp * 12472 for $2400 | 19:42 |
| kanzure | or 79 bp * 92918 for $4000 or 170 bp * 92918 for $6000 | 19:42 |
| kanzure | so 15 million bp for $6000. hmm. | 19:43 |
| kanzure | "CustomArray uses advanced CMOS semiconductor technology to enable it to synthesize thousands of oligonucleotides simultaneously. Each array contains thousands of individually addressable electrodes, which electrochemically synthesize a unique oligonucleotide at each electrode. The oligonucleotides are cleaved from the surface to create custom oligo pools." | 19:43 |
| kanzure | "Extremely competitive pricing -- as low as 0.04 cents per base." | 19:43 |
| kanzure | "On-board quality control systems" hmm looks like they put sensors on their microarrays | 19:44 |
| kanzure | "CustomArray's chips use electrochemical detritylation to control DNA synthesis. Electrochemical detritylation can be a much-more-efficient method to synthesize oligonucleotides than light-based synthesis processes. This means the oligonucleotides are of highest quality, and the sensitivity of the microarray is maximized. Since physical photolithographic masks or pre-built collections of oligos are NOT involved in the process, all probes ... | 19:45 |
| kanzure | ... can be easily changed without extra time or costs." | 19:45 |
| kanzure | pfft physical photolithography masks | 19:45 |
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| CaptHindsight | http://customarrayinc.com/faq_main.htm | 19:58 |
| kanzure | .wik tetrazole | 19:58 |
| yoleaux | "Tetrazoles are a class of synthetic organic heterocyclic compound, consisting of a 5-member ring of four nitrogen and one carbon atom (plus hydrogens). The simplest is tetrazole itself, CH2N4. They are unknown in nature." — https://en.wikipedia.org/wiki/Tetrazole | 19:58 |
| CaptHindsight | http://www.pnas.org/content/106/36/15219.full Photoelectrochemical synthesis of DNA microarrays | 20:02 |
| kanzure | also see http://diyhpl.us/~bryan/papers2/DNA/Photoelectromechanical%20synthesis%20of%20low-cost%20DNA%20microarrays%20-%20thesis%20-%202008.pdf | 20:04 |
| kanzure | filename is wrong heh | 20:04 |
| kanzure | more generally for photoremovable protecting groups, see http://diyhpl.us/~bryan/papers2/DNA/Photoremovable%20protecting%20groups%20in%20chemistry%20and%20biology%20-%20reaction%20mechanisms%20and%20efficiency.pdf | 20:04 |
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| CaptHindsight | kanzure: what are the costs of the chems used in photo deprotection vs the ones for inkjet? | 20:16 |
| ParahSailin_ | i wonder if you could cleave oligos from surface with electrochemistry too | 20:24 |
| ParahSailin_ | not clear here if all of the oligos need to be same length | 20:26 |
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| kanzure | CaptHindsight: it's probably on the azco biotech website | 20:37 |
| kanzure | ParahSailin_: for my purposes? nah different length is fine | 20:37 |
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| kanzure | ParahSailin_: also, if you're unable to cleave (chemically/electronically/whatever) then one option is just dropping in dna polymerase i think | 20:37 |
| ParahSailin_ | pretty much anyone wants different length-- certain array processes dictate that all oligos be the same length | 20:38 |
| ParahSailin_ | so if this one supports mixed lengths, thats just one more plus | 20:39 |
| kanzure | oh, why do the others require same length? .. no skips? | 20:39 |
| ParahSailin_ | affy's im pretty sure does, so does mycroarray | 20:40 |
| kanzure | i think one of the inkjet-related cleaning steps might be unskippable- like when blowing argon at everything. | 20:40 |
| kanzure | actually maybe the argon cleaning can be more specifically targeted | 20:40 |
| CaptHindsight | how long does each base take using Photoelectrochemical synthesis? | 20:41 |
| ParahSailin_ | does inkjet ever atomize liquid? | 20:41 |
| CaptHindsight | define atomize | 20:41 |
| CaptHindsight | what size drop? | 20:41 |
| ParahSailin_ | does it make a droplet that only hits where you want it without contaminating every cell in N radius | 20:42 |
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| kanzure | only hits where you want it | 20:42 |
| ParahSailin_ | that is a big concern with the lithography, be it mask or scanning laser | 20:42 |
| CaptHindsight | drop volume from the current Epson heads are 1.5pL | 20:42 |
| ParahSailin_ | so no molecules hit the wrong place? | 20:42 |
| kanzure | correct | 20:42 |
| ParahSailin_ | that seems unlikely that it doesnt aerosol at all | 20:43 |
| kanzure | it's pressurized | 20:43 |
| CaptHindsight | surface tension | 20:43 |
| ParahSailin_ | we've done some interesting experiments with aerosoling in liquid handling | 20:43 |
| ParahSailin_ | easily see 3% contamination in adjacent wells of a 384 well plate | 20:43 |
| ParahSailin_ | and thats at macro scale | 20:43 |
| kanzure | yeah but i bet you had humidity and stuff | 20:44 |
| ParahSailin_ | who knows | 20:44 |
| ParahSailin_ | maybe not a concern at all | 20:44 |
| ParahSailin_ | seems the electrochemistry is pretty battle tested | 20:44 |
| CaptHindsight | 20-28 seconds | 20:45 |
| CaptHindsight | Complete deprotection or detritylation was achieved without noticeable acid diffusion halos typically between 20–28 s | 20:49 |
| CaptHindsight | http://www.pnas.org/content/106/36/15219.full#F3 | 20:50 |
| CaptHindsight | this is a lot faster than inkjet | 20:51 |
| CaptHindsight | have to see the price of the chems | 20:51 |
| kanzure | inkjet can do more spots | 20:51 |
| kanzure | .title | 20:52 |
| yoleaux | Photoelectrochemical synthesis of DNA microarrays | 20:52 |
| kanzure | this is going to be limited by the size of the LCD | 20:52 |
| kanzure | inkjets can trivially do >100 million spots | 20:52 |
| CaptHindsight | apples and oranges | 20:52 |
| ParahSailin_ | optimize for number of spots before you can even do anything with 100 spot? | 20:53 |
| kanzure | 100 spots is useless | 20:53 |
| ParahSailin_ | 96k spots? | 20:53 |
| kanzure | significantly more useful | 20:54 |
| ParahSailin_ | 100 spots would not be useless if you had a good method of assembly | 20:55 |
| ParahSailin_ | id say that would be a viable product | 20:55 |
| ParahSailin_ | being able to make a plasmid in the 10-20kb range in one machine step | 20:56 |
| kanzure | each spot would be <100 bp, how are you getting 20 kb from 100 spots? | 20:57 |
| ParahSailin_ | azco will do up to 200bp in a spot | 20:57 |
| kanzure | i wouldn't trust yield to be that good yet- but if we can push it that high then sure... | 20:58 |
| ParahSailin_ | i dont know what the error rate is like, you might need to keep it below 100 for that reason, but 5-10kb is nothing to scoff at | 20:58 |
| kanzure | right | 20:58 |
| CaptHindsight | how many seconds per base using inkjet? | 21:01 |
| kanzure | don't know; hard to find. | 21:05 |
| CaptHindsight | looks like a few minutes from the sample program | 21:06 |
| CaptHindsight | http://customarrayinc.com/images/Array_Manufacture_Features.gif | 21:10 |
| CaptHindsight | not difficult to fab | 21:13 |
| CaptHindsight | this is the chemistry to look at | 21:14 |
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